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English Version

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mercoledì, agosto 1, 2007

A promising therapeutic agent

The CRM197(Cross-reacting material 197) pertains to a group of mutant diphtheria toxins obtained in the early 1970s from strain of Corynebacterium diphtheriae lysogenized with β-phages carrying a mutated tox gene.

It is completely non-toxic, shares the strong immunogenicity of the native molecule and has a unique ability to link heparin-binding epidermal growth factor (HB-EGF), the specific cell membrane receptor for diphtheria toxin (DT).

The CRM197 receptor is HB-EGF, a member of the EGF receptor. HB-EGF is overexpressed in the uterus during blastocyst implantation, in would healing, in many tumors and in cells of the atherosclerotic plaque.

The CRM197 has an activity to inhibit the binding of HB-EGF to EGF receptor by binding to an EGF-like domain in soluble and non-soluble (membrane-anchored) HB-EGF. A receptor binding domain in diphtheria toxin is involved in this binding.

The CRM197 is currently used as a carrier for infantile vaccines(see, in particular, Bartolozzi G, Rappuoli R.. I vaccini, UTET, 2001. pp 114, 164)

It often happens that it is the people in the country that you are born that prove deaf to your voice.

In fact, there is much truth in the old saying, “Nemo est propheta in patria”. Dr Silvio Buzzi is an unheeded prophet, both in the laboratory and in life. This is the story of someone who for 30 years, both as a scientist and as a man, has pursued a bold and powerful idea. It is an almost heretical idea for many within the great scientific community: to unleash against cancer the diphtheria toxin.

But “heresy” means “making one’s own choice”. Buzzi chose not to let himself be conditioned by conventional thinking. The thousand tales of desperate people who have turned up in Buzzi’s surgery, either by chance or through word-of-mouth, appear to prove him right and these tales are reinforced by the attention he has received from highly authoritative scientific journals in North America. It really does seem that the alien invader, the cancerous mass, has a low tolerance for the poison produced by the diphtheria bacterium. What was it that inspired this doctor from Ravenna to use a biological poison to contain the growth of tumours? “There was a phenomenon that always used to amaze me”, relates the 76 year old Dr Buzzi: “the hopeless cases, patients the doctors had given up on who inexplicably recovered after operations where nothing was done but cut open and sewn up again”.

Silvio Buzzi remembers his early years in Bologna, just freshly graduated and working in a Ravenna clinic, a converted block of flats. “Here, the Big Boss used to say to me, in relation to those strange (if temporary) recoveries of cancer patients: “Who knows …maybe the ‘breath of fresh air’ received by the viscera when the abdomen was opened up harmed the tumour in some mysterious way …”. Buzzi, however, never uses the expression “in some mysterious way”. “It’s true that there were only a few dozen of these peculiar cases reported in medical and scientific literature worldwide. But I really felt it was absolutely necessary for someone to make an effort to decipher them”. By now you know who that someone was.

A flash of insight came one morning: “I was in the operating theatre. People were getting ready to operate. The talcum powder, sprinkled in the rubber gloves to make them slide on, wafted in the air, forming a fine, visible haze under the glare of the lights. For a few minutes, the air around us was polluted. The tiny particles of talcum powder had contaminated it. And I had noticed it …”. The suspicion then arose that some micro-organism could pollute the operating room, could silently infect the surgical incisions.

In so doing, it could trigger an unforeseeable, hidden and positive immune response in the patient’s body … But the question was: could the regression of reliably-diagnosed cancerous masses derive from an accidental, unrecognised infection? This is the query that has animated Silvio Buzzi’s entire life.

While searching for the phantom, healing “germ”, he directed his attention one day to diphtheria. “It’s a serious, infectious disease, caused by the action of a toxin produced by a bacterium that is transmitted aerobically”, explains Buzzi. It is a powerful, toxic substance but one capable of evoking a huge response from the immune cells.

In fact, when someone is fighting diphtheria, there is an enormous increase in the number of white blood cells. Well, thought Buzzi, if that toxin is appropriately weakened, couldn’t it lead the body defence army against the anarchic cells of the enemy cancer?

These were questions never far from Buzzi’s thoughts over the years, even though his chosen career was that of a neurologist and psychiatrist.

However, from 1971, using whatever spare time life in his small province allowed him, in the narrow confines of the attic of his home converted into an animal laboratory and supported by the determination of his biologist wife, Luciana, he began a huge project using rats (and rabbits), injecting them with micro-doses of the diphtheria toxin. Tests, checks, analyses, confirmations … and something happened: the growth of solid tumours in the rodents treated with the poison was consistently blocked. It was an achievement that ended up in the (1973) Cancer Research journal. “From the halls of international medicine, we received only displays of curiosity”, recalls Buzzi. “There were no requests for closer collaboration. From Italian oncologists? An embarrassing silence. Our idea was too far removed from the preferred lines of research.”

His suspicions changed to certainty in the years to come when Doctor Silvio’s determined research became more refined. Now it involved the living reality of his patients. “I subsequently worked with a mutated diphtheria toxin, the CRM197. A “false” poison, which is a totally non-toxic substance but one just as capable of generating a robust immune response and linking to a specific target covering the surface of the tumour cells”.

What did Dr Buzzi find? “We gave the protein subcutaneously to a group of patients with advanced tumours and we then treated a number of other patients by injecting the toxin directly into the cancerous mass. Despite the poor general condition of most of the people involved, CRM197 had a very promising anti-cancer effect. Direct inoculation, on the other hand, clarified what phenomena were triggered off by the toxin in the malignant lesion: a massive influx of leucocytes and white blood cells. These are generators of enzymes and free radicals that end up damaging the cancerous cells”.

All this is documented: all the results were published (2004) in the journals Cancer Immunology and Immunotherapy and Therapy. It’s the final chapter in the exhausting adventure story of Buzzi’s endeavours. Or is it? Like all “prophets” unheeded in their country of origin, Buzzi and his staff were noted on the Internet by a team of Japanese scientists from three distinguished universities and have now been invited to participate in an extremely rigorous human study. “They have told me that they already have the ministerial funding. But we’ll now step aside”, declares the doctor with ironic bitterness: “we’ll hand over to our Japanese colleagues all our work in relation to the diphtheria toxin and CRM197 but we won’t be participating actively in the tests. A gift for foreigners … the price to pay in order to see my idea fully developed”.

Besides, the Buzzi family is tired: Silvio, Luciana, their children Annamaria, Giorgio and Silvia (children who have contributed to their father’s research: the first two as doctors and Silvia as a mathematical expert) have worked surrounded by general indifference (and light years away from the operations of university fiefdoms) to the point of physical and mental exhaustion.

We asked Buzzi: “What are you expecting from your work?” “I’m not looking for any limelight in television studios. That’s not where science is carried out. I embraced an idea. It is a possible further line of research in the daily battle against cancer. It was an intuition that, despite the material and economic limitations with which it has been developed, still generated some good: the official American journals that have welcomed my work but above all, the people who were given an inauspicious diagnosis who are still here to smile at me, 10-15 years after a medical sentence against which no appeal seemed possible”.

Buzzi does not caustically denounce academic plots and boycotts. He speaks with the gentlemanly detachment of a person who has long experienced the arduous task of living. Asked how many patients have you treated with “your” toxin, doctor, Buzzi answers, “Well, it’s not really “mine”. Do you want to know something? The Japanese were stunned to learn that none of us had ever thought about a patent. Instead, we published a detailed account of this new type of therapy which ultimately ended up preventing anyone from claiming ownership of the treatment!”

“How many patients?” “More than a thousand. But don’t ask me, for God’s sake, for survival rates and suchlike. I know perfectly well that my cases do not constitute a binding proof. “

The trouble is that experimental studies that allow one to evaluate the efficacy of a specific treatment in a particular population cost money. The Japanese have not had to work as hard as us but at least they have the means available to fully weigh up our therapeutic plan”. Buzzi feels he has reached the end of his personal scientific road. He adds: “I’ll continue to follow the progress of those few cases I still have in my care but my supplies of CRM197 (that the Italian branch of the famous Californian company, Chiron, donated to me) have now almost run out. And I’m not planning on buying up any more. At this point, I want to wait for a clear result from the Japanese tests”.

Two things have recently alleviated Dr Buzzi’s discontent (once again coming from abroad, as destiny would have it …): the decision taken by the American Association for Cancer Research to welcome him as one of their more active and authoritative members. Then there is the emotional email from an American housewife, Annette, in which she confirmed with her moving testimony Dr Silvio’s “crazy idea”. Her husband, Dave’s liver, lungs and tibia were riddled with metastases, stubbornly resistant to all chemotherapy treatment. He was doomed. And, as if this wasn’t enough, two months earlier the man had contracted an unidentified infection that had forced him to be admitted to hospital. Then, the “miracle” happened. Over time, the cancerous lesions, to the general amazement of the oncologists, melted away. In the laboratory report that had analysed his blood sample they discovered what the infection was, one word stood out: diphtheria.

Many years ago, diphtheria toxin (DT) showed antitumor activity in mice and in humans, but it was unclear whether this depended on the toxicity of the molecule only or on its strong inflammatory-immunological property as well. (Buzzi S., Cancer Res. 1982 May;42(5):2054-8). The same researchers, to deal with this open question, planned to treat a group of cancer patients with cross-reacting material 197 (CRM197).

CRM197 is a nontoxic mutant of DT that shares the immunological properties of the native molecule and its ability to bind to heparin-binding epidermal growth factor (HB-EGF), the specific cell-membrane receptor for DT that is often overexpressed in cancer. 25 patients with various advanced tumors who were refractory to standard therapies (23 subjects) or had refused, in whole or in part, conventional therapies (2 subjects) were treated with CRM197 injected subcutaneously in the abdominal wall, on alternate days, for 6 days. Three different dosages (1.7, 2.6, or 3.5 mg/day) were used according to the patient’s degree of immunological reactivity to DT/CRM197 (none, moderate, or high).After the first administration of CRM197, a significant increase in the number of circulating neutrophils and in the serum level of TNF-alpha was detected. Toxicities were minimal. Only patients with delayed-type hypersensitivity to DT/CRM197 had irritating skin reactions in the injection sites and a flu-like syndrome with fever. Pharmacokinetics showed a mean peak concentration (12.7 ng/ml) 12 h after the first injection and a mean half-life of 18.1 h. There were two complete and one partial responses (metastatic breast carcinoma, neuroblastoma, and metastatic breast carcinoma) lasting 4, 45+, and 15 months, respectively. Six cases of stable disease, lasting from 1 to 15 months, were also recorded. CRM197 injected subcutaneously elicited an inflammatory-immunological reaction, caused tolerable toxicities, was absorbed to a good extent into the circulatory system, and exerted some degree of biological antitumor activity. A possible role of neutrophils and TNF-alpha in the mode of action of the molecule has been hypothesized, but not yet confirmed. This interesting molecule is currently under investigation in a phase II clinical trial held in Japan by Prof. Mekada and collaborators. Results are expected impatiently in less than a year. The rational under the antitumor activity of this molecule is still to be fully elucidated. Anyway, it seems to hold the noteworthy property of reawaken the host immune system, often dormant or inefficient in cancer patients. An excerpt from Cancer Immunol Immunother. 2004 Nov;53(11):1041-8.